We have continued our studies of chromatin structure in the neighborhood of expressed genes. The globin gene family in chicken erythroid cells serves as a model system in which it is possible to study the mechanisms associated with regulation of the cluster and individual members of the family during erythroid development. We have focused attention on the 1.2 kb insulator DNA sequence at the 5' end of the chicken beta-globin locus, and elements upstream of it. This insulator is capable both of blocking the influence of outside enhancers and of preventing the encroachment of condensed chromatin that might shut down expression of the entire region. We have shown previously that enhancer blocking activity is associated with binding of a single protein, CTCF, to a site within the enhancer. The insulator also has the separate ability to protect against position effects reporter genes that are stably transfected into cell lines or animals, serving as a boundary against encroachment of condensed chromatin. We found that this protective ability is present in a ?core' element, 250 bp long, from within the 1.2 kb insulator, and that deletion of subregions within the core that contain the CTCF site do not affect activity. However four other subregions corresponding to binding sites for nuclear proteins are important for position effect protection (boundary function). We showed that one of these binding sites is specifically responsible for maintaining a high level of histone acetylation and methylation at sites associated with gene activation. This site binds a heterodimer of the proteins USF1and USF2. These results are consistent with a model we have proposed in which barrier function is connected with multiple histone modifications in the neighborhood of the insulator. In an effort to understand how the USF proteins mediate this action, we have investigated what co-factors are recruited by USF1, both by chromatin immunoprecipitation in vivo and by co-precipitation in vitro. We have shown that the arginine methyl transferase, PRMT1, is recruited to the insulator, and that down regulation of PRMT1 by RNAi methods results in loss not only of histone H4 arg 3 methylation, but also in widespread loss of H3 and H4 acetylation over the genome. Now we have directly identified proteins interacting with USF1 by co-immunopurification with tagged USF1. When combined with gel filtration studies designed to detect stable complexes, this revealed the presence of two distinct complexes, one of which contained PRMT1 and the HATs PCAF and SRC-1. Consistent with its interaction with PRMT1, siRNA downregulation of USF1 resulted in localized loss of H4R3 methylation at the insulator; other histone modifications associated with euchromatin are also lost locally. Significantly, increases in H3K27 methylation, associated with heterochromatinization, spread downstream of the insulator when USF1 is depleted. This is consistent with a model in which the insulator normally works as a barrier by maintaining a local environment of active chromatin. [unreadable] [unreadable] We have carried on studies of the histone variant H3.3 in an avian erythroid cell line. H3.3 is incorporated into chromatin during interphase, whereas the predominant variant, H3, is incorporated only during S phase. H3.3 has been reported to be deposited predominantly downstream of active promoters. We have studied the distribution of H3.3 over the genes in and near the a-globin locus, as well as elsewhere in the genome. A wide variety of patterns was observed, notably including marked incorporation over upstream regulatory regions such as enhancers and locus control regions. In the case of FR (Folate Receptor) and VEGFD (Vascular endothelial growth factor D) in which incorporation is confined to upstream regions, the presence of exogenous H3 results in reduced expression, while H3.3 stimulates expression. This suggests that H3.3 may play an active role in regulation of gene expression. Studies are now being extended to other variants. [unreadable] [unreadable] As an extension of our earlier interest in chromatin structure and epigenetic regulation at the Igf2/H19 locus, we have ubdertaken similar studies in human islets, as well as in cells derived from other tissues, of the nearby insulin gene. We have also begun a collaboration with Drs. Marvin C. Gershengorn and Bruce Raaka, NIDDK, to carry out similar analyses of human Islet-derived Precursor Cells that have been induced to undergo an epithelial to mesenchymal transition. [unreadable] [unreadable] We continue a collaboration with Drs. David Fear and Hannah Gould (King?s College, London) and Dr. Martin Gellert, NIDDK, to examine the chromatin structure in the neighborhood of the germ line genes in the human immunoglobulin H chain locus. Our earlier studies had found that individual cells contain transcripts from multiple germ line genes, showing that chromatin structure over all the genes is open, and that structural changes per se could not be the basis of selctivity in class switch recombination. We are now beginning to measure chromatin structure and DNA methylation patterns over the locus before and during class switching.